重组人肿瘤坏死因子α对人脂肪基质细胞体外成骨向分化的影响

Recombinant human tumor necrosis factor-alpha promotes human adipose-derived stromal cells transforming into osteoblast in vitro

目的:探讨一定浓度的重组人肿瘤坏死因子α(recombinant human tumor necrosis factor alpha,rhTNF-α)对人脂肪基质细胞(human adipose-derived stromal cells,hASCs)体外成骨向分化的影响。方法:采用第4代hASCs,根据培养基的不同分为4组:(1)基础培养[DMEM+10%FBS(体积分数)+100 U/mL青霉素+100 mg/L链霉素];(2)基础培养+10μg/L rhTNF-α;(3)成骨向诱导培养(基础培养+100 nmol/L地塞米松+50μmol/L维生素C+10 mmol/Lβ-甘油磷酸);(4)成骨向诱导培养+10μg/L rhTNF-α。各组每3天更换相应培养基。在第3、7、14天和第21天进行碱性磷酸酶(alkaline phosphatase,ALP)定量检测;在第14和21天进行钙结节染色和矿化沉积定量检测;并用反转录PCR检测成骨向诱导组核心结合因子α1(core-binding factorα1,Cbfa1)、Osterix(Osx)和骨钙素(osteocalcin,OC)等成骨相关基因的表达;在成骨向诱导的第3天,用实时定量PCR检测rhTNF-α对hASCs成骨相关基因表达的影响。结果:对碱性磷酸酶定量检测,在成骨向诱导的第14天(3.527±0.415 vs.2.345±0.354,P<0.01)和第21天(3.106±0.105 vs.2.442±0.163,P<0.01),10μg/L的rhTNF-α可以促进其表达;同样对于矿化沉积检测,在成骨向诱导的第14天(2.896±0.173 vs.0.679±0.173,P<0.01)和第21天(2.231±0.233vs.1.729±0.229,P<0.01),10μg/L的rhTNF-α也促进其表达;反转录PCR和实时定量PCR也表明rhTNF-α可以促进Cbfa1,Osx和OC的表达。结论:10μg/L的rhTNF-α可以促进成骨向诱导的hASCs体外成骨向分化,并可以促进成骨相关基因Cbfa1、Osx和OC的表达。

Objective： To investigate the effect （rhTNF-α ） on the osteogenesis potential of the （hASCs） in vitro. Methods： hASCs at passage conditions ： basal medium [BM, DMEM ＋ 10% of recombinant human tumor necrosis factor alpha osteo-induced human adipose-derived stromal cells 4 were divided into four groups according to culturing FBS ＋ antibiotics], BM with 10 μg,/L rhTNF-α, osteogenic medium （OM, BM ＋ dexamethasone ＋ L-ascorbate ＋ β-glycerophosphate） and OM with 10 μg/L rhTNF-α. On days 3, 7, 14 and 21, alkaline phosphatase （ALP） activities were examined. On days 14 and 21, the staining and quantitation of calcium deposition were performed. For the cells under osteogenic induction, osteoblast- related genes, such as core-binding factor oil （ Cbfal ）, Osterix （Osx） and osteocalcin （OC） were analyzed with reverse transcription PCR on days 3, 7, 14, and 21, and real time PCR was performed to confirm the effect of rhTNF-α on genes expression on day 3 . Results： rhTNF-apromoted ALP activities of induced hASCs on day 14 （3.527 ± 0.415 vs. 2. 345± 0. 354, P 〈0.01 ） and on day 21 （3. 106±0. 105 vs. 2. 442 ± 0. 163 ,P 〈0.01 ） and promoted calcium deposition of induced hASCs on day 14 （2.896± 0.173 vs. 0.679 ± 0.173,P〈0.01） and on day 21 （2. 231±0. 233 vs. 1. 729± 0. 229, P 〈 0.01）. RT-PCR and Real-time PCR assays showed that rhTNF-α augmented the expression＇of Cbfal, Osx and OC of these cells. Conclusion： The findings indi- cate that 10 μg/L rhTNF-α can promote the osteogenic potential of osteogenetically induced hASCs in vitro.